Methods for the treatment or prevention of systemic sclerosis

ABSTRACT

The disclosure is in the field of molecular immunology, more in particular, in the field of the prevention or treatment of autoimmune diseases, more in particular, systemic sclerosis or scleroderma. The disclosure is based on the observation that SSC patients have an elevated plasma level of CXCL4. This was found to contribute to the pathogenesis of SSc, in particular, fibrosis. When CXCL4 was neutralized in in vitro experiments, the fibrotic effects could be neutralized. This led us to conclude that SSc may be cured by reducing the plasma level of CXCL4. The disclosure, therefore, relates to a method for treatment or prevention of fibrosis in patients with scleroderma, wherein the plasma level of CXCL4 is reduced.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/471,968, filed Aug. 28, 2014, pending, which is a continuation ofU.S. patent application Ser. No. 13/928,557, filed Jun. 27, 2013, nowU.S. Pat. No. 8,920,802, issued Dec. 30, 2014, which is a continuationof U.S. patent application Ser. No. 13/138,608, filed Sep. 8, 2011, nowU.S. Pat. No. 8,486,407, issued Jul. 16, 2013, which application is anational phase entry under 35 U.S.C. § 371 of international PatentApplication PCT/EP2010/052919, filed Mar. 8, 2010, published in Englishas International Patent Publication WO 2010/102983 A1 on Sep. 16, 2010,which claims the benefit under Article 8 of the Patent CooperationTreaty to European Patent Application Serial No. 09154582.2, filed Mar.8, 2009.

TECHNICAL FIELD

The disclosure is in the field of molecular immunology, more inparticular, in the field of the prevention or treatment of autoimmunediseases, more in particular, systemic sclerosis or scleroderma.

BACKGROUND

Scleroderma or Systemic Sclerosis (SSc) is an autoimmune connectivetissue disorder of unknown cause characterized by microvascular injury,excessive fibrosis of the skin, and distinctive visceral involvementincluding the heart, lung, kidneys and gastrointestinal tract.Scleroderma is a progressive condition in which fibrous tissue growsabnormally, causing the skin to thicken and harden, often disfiguringand disabling patients. It affects between 5,000-10,000 new personsannually (USA) and is associated with a high morbidity and a poorprognosis. Overall, scleroderma affects an estimated 300,000 Americans,most of them women.

Forty percent of all scleroderma patients develop at least moderaterestrictive lung disease. A high proportion of scleroderma patients (80percent) develop lung involvement, either interstitial lung diseaseand/or pulmonary hypertension, which are the leading causes of death dueto scleroderma. The mortality rate in scleroderma patients with severerestrictive lung disease is about 30 percent within 10 years of onset.

The statistics underscore the need for effective treatment, preferablyat an early stage in the illness, to prevent progression to severeinterstitial lung disease. SSc is treated with oral medications to haltthe progression of disease. These drugs include cyclophosphamide,high-dose prednisolone or even stem cell transplantation, all havingsevere side effects. In SSc, drugs that improve circulation, promotegastrointestinal function, preserve kidney function, and control highblood pressure are also given. Cyclophosphamide is an anti-cancer drugthat suppresses the immune system. This is the first drug that has beenproven to alleviate the most devastating effects of SSc. Patients takingcyclophosphamide had a significant improvement in lung function and areduction in breathlessness.

Although many research groups work on the unraveling of SScpathogenesis, to date, the exact pathways underlying the pathogenesis ofSSc remain unknown. Currently, most of the research is focused on theunderlying pathways that cause fibroblast activation. A deregulatedgrowth and activation of fibroblasts has often suggested to beimplicated in SSc. However, a mode of action that is responsible forthis ongoing activation of fibroblasts has not been identified.

DISCLOSURE

The disclosure is based on the observation that SSC patients have anelevated plasma level of CXCL4. This was found to contribute to thepathogenesis of SSc, in particular, fibrosis. When CXCL4 was neutralizedin in vitro experiments, the fibrotic effects could be neutralized. Thisled to the conclusion that SSc may be cured by reducing the plasma levelof CXCL4. The disclosure, therefore, relates to a method for treatmentor prevention of fibrosis in patients with scleroderma, wherein theplasma level of CXCL4 is reduced.

It was also found that CXCL4 was produced by pDCs in the bloodcirculation of patients with SSc. A method as described above is,therefore, preferably performed by depleting pDCs from the circulationof a patient with scleroderma.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Increased frequency of plasmacytoid DCs in SSc patients. Usingthe DC enumeration kit (Miltenyi Biotec), SSc patients showed a clearlyincreased frequency of plasmacytoid DCs (pDCs, BDCA2+), whereas thefrequency of myeloid DCs (mDCs, BDCA1+ or BDCA3+) was similarlydistributed among groups. Patients with diffuse cutaneous SSc especiallyhad a markedly raised frequency of pDCs that was most marked in thosehaving a disease duration shorter than 2 years.

FIG. 2: The increased frequency of pDCs is most pronounced in diffuseSSc. Using magnetic bead isolation of pDCs (BDCA4+ cells), it is clearthat patients with diffuse cutaneous SSc have a significantly increasedfrequency of pDCs compared to those having the limited cutaneousphenotype. In turn, patients having early diffuse (<2 years of diseaseduration) SSc have significantly higher frequencies of circulating pDCsthan those with late diffuse disease (>3 years of disease duration).

FIG. 3: CXCL4 in pDC supernatant and plasma from SSc patients ismarkedly increased. CXCL4 levels were measured using ELISA techniques inpDC supernatant (Panel A) derived from healthy controls and patientswith limited, late diffuse and early diffuse SSc. CXCL4 levels areincreased in all SSc patients but more pronounced in patients havingdiffuse SSc, especially those having early diffuse cutaneous SSc. PanelB displays the CXCL4 levels in the plasma of healthy controls and SScpatients. CXCL4 levels in both pDC supernatant as well as plasma closelyreflect the frequency of pDCs in the circulation.

FIG. 4: CXCL4 directly stimulates fibroblasts. Although unstimulatedskin fibroblasts do not express high levels of COMP, co-incubation withSSc plasma resulted in a clear up-regulation of COMP expression that wasabrogated by heparinase, a natural CXCL4 antagonist. In contrast, plasmafrom healthy controls did not induce the up-regulation of COMP.

FIG. 5: The addition of CXCL12 to SSc pDCs induces a self-perpetuatingactivation loop. Although the IL-6 production by pDCs from healthycontrols could only be induced by the co-incubation with CXCL4+CXCL12,SSc pDCs produced large amounts of IL-6 upon the addition of CXCL12.This effect could be fully abrogated by the addition of heparinaseunderscoring the CXCL4 dependency in this system. The addition of CXCL4only did not have a significant effect, suggesting the need for CXCL12in the CXCL4-dependent pDC activation loop.

FIG. 6: pDCs from SSc patients secrete higher levels of Type I IFNs uponTLR-mediated stimulation. Upon stimulation of pDCs with ligands forTLR2, TLR3, TLR4 and TLR9, pDCs from SSc patients produce significantlymore type I IFN compared with healthy controls. More specifically, SScpatients with diffuse cutaneous disease showed a markedly increasedsecretion of type I IFN.

FIG. 7: The augmented TLR response by pDC in SSc is abrogated byneutralization of PF4. The administration of heparinase, a natural CXCL4antagonist, fully abrogates the augmented TLR response as observed inSSc patients.

DETAILED DESCRIPTION

It was found that the frequency of plasmacytoid DCs (pDCs) is markedlyincreased in SSc patients. This is illustrated in FIG. 1. In particular,the frequency of pDCs was increased in patients with the diffusecutaneous subtype of SSc. The frequency of pDCs was highest in patientssuffering from early diffuse SSc. The number of pDCs in individual SScpatients, as compared to normal healthy controls, is shown in FIG. 2.

It was also found that plasma from SSc patients contains high levels ofCXCL4. CXCL4 is a chemokine described to bind to CXCR3, morespecifically, the splice variant CXC3b, which is in contrast to CXLC9,CXCL10 and CXCL11 that bind to CXCR3a (Lasagni et al., J. ExperimentalMedicine 2003). CXCL4 is known to induce apoptosis in endothelial cellsleading to endothelial cell death. In addition, CXCL4 is known to havepotent anti-angiogenic properties and known to induce fibroblastactivation via activation of monocyte-derived dendritic cells.

CXCL4 was measured in a standard ELISA assay and the results areillustrated in FIG. 3, Panel A. It is shown that CXCL4 was overproducedby pDCs of SSc patients since elevated levels of CXCL4 could also bedetected in the supernatant of pDCs isolated from early diffuse SScpatients. This is illustrated in FIG. 3, Panel B. It was noted that theproduction of CXCL4 in pDC supernatant closely mirrored the frequency ofpDCs in plasma. Since CXCL4 was originally identified as a sole plateletactivation marker, the possibility was excluded that plateletscontaminated the pDCs supernatants by measuring the β-TG content, whichwas negligible.

Hence, it was concluded that CXCL4 is over-produced by pDCs in patientswith SSc. This was confirmed in a whole proteome analysis usingSELDI-TOF. Analysis of pDC revealed that SSc pDCs were solely committedto produce a few proteins that could be identified as CXCL4 orCXCL4-precursor molecules.

It was also shown that CXCL4 plays a role in fibroblast activation inSSc patients. To that end, the expression of known fibroblast activationmarkers TGFβR, COMP and αSMA in healthy fibroblasts co-incubated withSSc plasma was measured. It was found that SSc plasma induced theexpression of these markers for fibroblast activation significantly andin a dose-dependent manner (FIG. 4). In addition, the plasma fromhealthy donors had no effect and the effect of SSc plasma was abrogatedusing heparinase, a natural antagonist of CXCL4. It was concluded thatan elevated level of pDCs in Ssc patients contributes to the fibroticprocess.

CXCL4 on its own was found to be unable to stimulate pDCs to producepro-inflammatory molecules; neither pDCs from normal individuals norpDCs from SSc patients produced pro-inflammatory molecules. Only in thepresence of CXCL12-specific stimulation of pDCs derived from SScpatients could be observed. This is illustrated in FIG. 6. Therein, theproduction of pro-inflammatory molecules was measured in the presence ofpDCs from healthy individuals as compared to pDCs isolated from SScpatients.

pDCs from healthy controls were incubated with plasma from SSc patientsand healthy controls, after which the production of pro-inflammatorymolecules was measured. The addition of plasma from SSc patients andhealthy controls had no effect. pDCs from healthy individuals wereincubated, as well as from SSc patients with SSc plasma and healthycontrol plasma, with and without the presence of CXCL12. It was foundthat only the combination of SSc plasma and CXCL12 led to asignificantly increased level of IL-6 and IL-12. To assure that thiseffect was due to the “auto-activation” of pDCs by endogenous CXCL4,CXCL4 was added to the cultures of pDCs from healthy individuals withhealthy control serum and CXCL12. The addition of CXCL4 to theseexperiments induced IL-6 and IL-12 secretion to the same levels as thecombination of SSc plasma and CXCL12 (FIG. 5). In addition, the additionof heparinase fully abrogated the effect of SSc plasma and CXCL12 onpDCs.

Without wanting to be bound by theory, it was hypothesized that CXCL12(also known as SDF-1) binds to CXCR4, thereby initiatingchemo-attractive properties aimed at the recruitment of pDC toward thesite of inflammation. CXCL12 is known to be highly presented in the skinof SSc patients and likely to be produced by local fibroblasts. Insupport of this hypothesis, it is mentioned that monocyte-derived DCswere found to be insensitive to CXCL4 unless CXCL12 is present. It iscurrently unknown how CXCL12 sensitizes DCs to the effect of CXCL4(Cipriani et al., Arthritis Rheum. 2006 Sep., 54(9):3022-33).

It is known that pDCs are stimulated mostly via toll-like receptor 9(TLR9), via which they mainly make type I interferons (IFNs). As such,pDCs are considered the main producers of this cytokine.

Challenged by the question “what induces the increased CXCL4 productionby pDCs,” the role of toll-like receptors (TLR) was investigated. Theseexperiments showed that TLR3-, TLR7/8- and TLR9-mediated stimulationinduced CXCL4 secretion by pDCs. Interestingly, DCs from SSc patientswere found to produce much more IFNs and IL6 than those from healthycontrols (FIG. 6). A clearly increased secretion of IFNα in lcSScpatients, ldSSc patients and edSSc patients was observed.

This shows that the potentiated TLR response by pDCs is caused by aCXCL4-dependent TLR augmentation. In support of this, neutralization ofCXCL4 led to a full abrogation of this TLR augmentation, showing thatCXL4 underlies this phenomenon (FIG. 7).

To further substantiate these findings, pDCs from healthy controls wereincubated with CXCL12, CXCL4 or its combination dose dependently. Theseexperiments demonstrated that CXCL4 in combination with CXCL12 led to anaugmentation of the TLR response.

In conclusion, these observations show that CXCL4 (in conjunction withCXCL12) plays a central role in the pathogenesis of SSc. The mechanismfor that is auto-activation of pDCs resulting in a potentiated TLRresponse and further CXCL4 production.

Patients with SSc may, therefore, benefit from methods that remove CXCL4and/or pDCs, for instance, from the blood circulation. That may beaccomplished in a number of ways, which are known in the art per se.

Patients with scleroderma may benefit from a treatment wherein theeffects of the CXCL4 produced in excess are neutralized or counteracted,for instance, by removing CXCL4 from circulation. In the alternative,CXCL4 may be prevented from binding to its receptor. The disclosure,therefore, relates to a compound capable of binding to CXCL4 for use inthe treatment or prevention of fibrosis in patients with scleroderma.

Compounds capable of binding to CXCL4 may be compounds capable ofbinding to CXCL4 under physiological conditions.

Compounds capable of binding to CXCL4 per se are known in the art. Forinstance, fragments of the receptor or fragments of CXCL4 will besuitable compounds. In a preferred embodiment, specific antibodies maybe employed in order to remove CXCL4, for instance, from thecirculation. This may be accomplished by plasmapheresis.

Antibodies against CXCL4 are available in the art. They have beendescribed, for instance, in Vandercapellen et al., J. Leukocyte Biol.82, 1519 (2007), and are commercially available from R&D Systems, 614McKinley Place NE, Minneapolis Minn. 55413.

Given the teachings of this disclosure, it will be within the routineskills of a person skilled in the art to design and develop materialsand methods for removing or counteracting the effects of CXCL4 and/orDCs such that patients with SSc may benefit from these materials andmethods.

For that purpose, CXCL4 antagonists may be advantageously employed.Antagonists for CXCL4 are known to the skilled person. As an example,heparinase is disclosed and exemplified herein.

In another embodiment, the disclosure also relates to a compound capableof interfering with the in vivo production of CXCL4 for treatment orprevention of fibrosis in patients with scleroderma. Compounds capableof interfering with the in vivo production of CXCL4 per se are known inthe art. The disclosure, therefore, also relates to a method fortreatment or prevention of fibrosis in patients with scleroderma byinterfering with the in vivo production of CXCL4. Such methods mayencompass interference at the level of transcription or translation ofCXCL4. Such methods are known in the art and may now advantageously beused in the treatment of scleroderma.

A reduction in the level of CXCL4 in a patient's body may beaccomplished by a therapy in vivo. Alternatively, patients with SSc mayalso benefit from in vitro methods that reduce the level of CXCL4 inblood. Such may be done directly by applying a compound capable ofbinding to CXCL4 and contacting that compound with blood obtained from apatient with SSc. It may also be accomplished by reducing the number ofDC that produce the CXCL4 in patients with scleroderma. This may also beaccomplished in an in vitro method wherein the previously isolated bloodor plasma from a patient with scleroderma is contacted with a compoundor device capable of reducing or depleting the number of pDCs insolution. The disclosure, therefore, also relates to an in vitro methodfor reducing the level of CXCL4 and/or the number of pDCs in bloodobtained from a patient with scleroderma. In more detail, the disclosurerelates to an in vitro method for reducing the level of CXCL4 and/or thenumber of pDCs in a sample obtained from a patient with scleroderma,comprising the steps of:

-   -   a. providing the sample obtained from a patient with scleroderma    -   b. contacting the sample with a compound capable of binding to        CXCL4    -   c. allowing the compound to bind to CXCL4 in order to form a        complex    -   d. removing the complex from the sample, thereby reducing the        level of CXCL4 and/or the number of pDCs.

Devices capable of reducing the level of CXCL4 and/or pDCs in blood areknown in the art. Preferably, CXCL4 and/or pDCs are removed fromcirculation, for instance, using a commercially available techniqueprovided by Miltenyi (Jongbloed et al., Arthritis Res. Ther. 2006,8(1):R15).

Miltenyi produces and markets such a device that may be advantageouslyused in the new treatment according to the disclosure. Hence, thedisclosure relates to a device capable of reducing the level of CXCL4and/or pDCs in blood for the treatment or prevention of fibrosis inpatients with scleroderma.

Alternatively, a method according to the disclosure may also comprise astep wherein the binding of CXCL4 to its receptor is blocked orotherwise prevented or down-regulated or decreased. The disclosure,therefore, also provides a method for treatment or prevention offibrosis in patients with scleroderma by interfering with the binding ofCXCL4 to its receptor.

EXAMPLES Example 1: Isolation of PBMCs, Plasmacytoid DCs and PhenotypicCharacterization

PBMCs were isolated from heparinized venous blood (100 ml) by usingdensity-gradient centrifugation over FICOLL-PAQUE® (AmershamBioscience). Next, plasmacytoid DCs (pDCs) were isolated using the BDCA4magnetic isolation kit from Miltenyi Biotec according to themanufacturer's protocol. All procedures were carried out on ice andafter the isolation of pDCs, these cells were counted usingcounterchambers and directly transferred to RPMI medium without anyadditions in a concentration of 1 million cells/ml. After pelleting, thesupernatant was collected and stored at −80° C. until further analysis.The cells were then resuspended in RPMI supplemented with 2 nML-glutamine, 100 U/μL/ml penicillin/streptomycin (Life Technologies),and 10% FCS (BioWhittaker), after which cells were brought into culturefor further analysis. Directly after isolation, a part of the BDCA4+cells were resuspended in FACS buffer and were analyzed for theexpression of BDCA1, BDCA2, BDCA3, CD80, CD86, MHC and FcγRs applyingstandard flow cytometry protocols. During the first experiments, thepDCs diamond kit from Miltenyi Biotec was exploited to replicate thedata having so-called “untouched pDCs.” In all experiments, direct DCenumeration was performed using the DC enumeration kit from MiltenyiBiotec according to the manufacturer's protocol.

Example 2: Measurement of inflammatory mediators

Supernatant from unstimulated pDCs was stored after 1 and 24 hours ofculture. In addition, pDCs were stimulated with ligands specific fortoll-like receptor 2 (TLR2, pam3Cys), TLR3 (Poly-IC), TLR4 (LPS),TLR7/TLR8 (R848) and TLR9 (CPG) for 24 hours, after which, supernatantwas collected. In all these supernatants, the levels of multipleinflammatory mediators (IL-8, TNF, IFNγ, IFNα, MCP-1, MIP1β, IL-6,IL-1β, IL-12. IL-23, IL-17F, IL-17A) were measured using Luminextechnology as previously described (Roelofs et al., Arthritis Rheum.2005 Aug., 52(8):2313-22; Roelofs et al., Ann. Rheum. Dis. 2009 Sep.,68(9):1486-93, Epub 2008 Sep. 2; Wenink et al., J. Immunol, 2009 Oct. 1,183(7):4509-20, Epub 2009 Sep. 4). To study the full proteome in pDCs,the 1-hour spontaneous supernatants were used for analysis in theSELDI-TOF. Exploiting this technique, 40 supernatants were testedoriginating from healthy controls (n=10) and SSc patients having limitedcutaneous (n=10), late diffuse (n=10) and early diffuse (n=10) diseaseas previously described (Radstake et al., PLos ONE 2009). The expressionof CXCL4 and β-TG was measured by commercially available ELISAs usedfollowing the manufacturer's protocols.

Example 3: Stimulation of Plasmacytoid DCs and Investigating theSelf-Perpetuating Loop

Since the literature suggests an important role for CXCL12 in theCXCL4-mediated activation of pDCs, the potential role of this loop inSSc was investigated (van der Vliet et al., J. Experimental Medicine2003 Sep. 1, 198(5):823-3). To this aim, pDCs from healthy controls andSSc patients were first co-cultured with combinations of CXCL4 and/orCXCL12. During these experiments, pDCs were cultured with RPMI only,RPMI/CXCL12, RPMI/CXCL12/CXCL4 and RPMI/CXCL12/heparinase. In addition,the effect of CXCL4 on the TLR pathways was investigated bypre-incubation of pDCs with CXCL4 or the combination CXCL4/CXCL12, afterwhich, pDCs were consecutively stimulated with TLR ligands. After 24hours of incubation, the production of IFNα, IL-12 and IL-6 was measuredby Luminex.

Example 4: CXCL4-Mediated Activation of Fibroblasts

Skin fibroblasts were isolated from healthy skin biopsies and culturedaccording to standardized protocol as described previously (Farina, Ann.Rheum. Dis. 2009 Mar., 68(3):435-41). When confluent, fibroblasts werereplated and cultured for an additional 24 hours to allow attachment tothe culture plates. Subsequently, fibroblasts were incubated withcombinations of plasma from healthy controls and SSc patients, plasmaand heparinase and CXCL4/CXC12. After 48 hours of culture, theexpression of COMP was measured by real-time PCR as previously described(Farina, Ann. Rheum. Dis. 2009 Mar., 68(3):435-41).

1.-15. (canceled)
 16. A method for treating a subject diagnosed assuffering from diffuse cutaneous systemic sclerosis, the methodcomprising: depleting plasmacytoid dendritic cells in the subject,thereby treating diffuse cutaneous systemic sclerosis in the subject.